Expression And Mechanism Of CDCA3 In Hypopharyngeal Squamous Cell Carcinoma

Background:About 3%to 5%of head and neck squamous cell carcinoma cases are hypopharyngeal squamous cell carcinoma(HSCC).Due to the lack of typical clinical symptoms in the early stage of the disease,more than 70%of the patients with hypopharyngeal cancer are in stages Ⅲ and Ⅳ at the initial clinical visit.HSCC is highly invasive and prone to submucosal spread in the early stages of the disease;moreover,the risk of cervical lymph node metastasis is very high.HSCC is highly malignant and has a poor prognosis;therefore,treatment is very challenging and often includes surgery,chemotherapy,radiotherapy,or a combination of these.However,the 5-year overall survival rate of HSCC is only approximately 15-45%and has not improved much in the past few decades.Therefore,there is an urgent need to further study the molecular mechanism(s)of HSCC progression and identify new and effective therapeutic targets.Disruption of the cell cycle plays a vital role in the development and progression of tumors.Cell division cycle-related protein 3(CDCA3),also known as TOM-1,is necessary for mitoticentry.CDCA3 of human beings is located on chromosome 12p12.It has 268 amino acids with the molecular weight of 29 kDa.CDCA3 is a member of the F-box protein family,which contains an F-box motif.As part of the Skp1-Cullin RING-F-box(SCF)ubiquitin ligase(E3)complex,CDCA3 effectively degrades the endogenous cell cycle inhibitor,thus playing a vital role in cell cycle regulation.SCF E3 ligases regulate many biological processes by regulating numerous short-acting proteins,such as cell cycle-regulating proteins,transcription factors and signal transduction molecules.More and more researches show that CDCA3 expression increases in esophageal cancer,liver cancer,gastric cancer,colorectal cancer,oral squamous cell carcinoma,non-small cell lung cancer and prostate cancer cells,which may be related to the occurrence and development of malignant tumors.However,the biological role of CDCA3 and its potential mechanism in HSCC remain undetermined.This is the first study about the expression and significance of CDCA3 in hypopharyngeal squamous cell carcinoma.The study was carried out from the following aspects:firstly,the expression of CDCA3 in hypopharyngeal squamous cell carcinoma and adjacent tissues of clinical specimens was detected to analyze the relationship between its expression level and clinicopathological features and prognosis of patients with hypopharyngeal squamous cell carcinoma,and then a lentivirus silenced CDCA3 cell line was constructed to study its effect on cell phenotype in vitro.Finally,the molecular mechanism of CDCA3 in HSCC was explored by using second-generation sequencing and bioinformatics analysis technology,all the efforts were to make it possible to develop new anti-cancer therapies for hypopharyngeal squamous cell carcinoma.Part 1 The expression and clinical significance of CDCA3 in hypopharyngeal squamous cell carcinomaObjectives:We detected the expression of CDCA3 in HSCC tissues and normal tissues adjacent to the cancer,and then analyzed the relationship between its expression and clinicopathological characteristics and prognosis of patients with HSCC.Methods:1.Firstly,qRT-PCR and Western blot analysis were used to detect the expression of CDCA3 mRNA and protein in HSCC tissues and adjacent normal tissues,and then the expression of CDCA3 protein in 80 HSCC patients was detected by immunohistochemistry(IHC).2.The relationship between the expression of CDCA3 and the clinicopathological features of patients with HSCC were analyzed.Kaplan-Meier survival curve analysis was used to evaluate the correlation between the expression of CDCA3 and the prognosis of patients with HSCC.Cox proportional hazard regression model was used to analyze the independent prognostic factors affecting the overall survival of patients with HSCC.Results:1.The results of qRT-PC,Western blot and IHC showed that the relative expression levels of mRNA and protein of CDCA3 in hypopharyngeal squamous cell carcinoma were higher than those in adjacent normal mucosa(P<0.05).2.The protein expression level of CDCA3 was related to the clinicopathological features such as tumor T staging,lymph node metastasis and tumor TNM staging(P<0.05).3.Kaplan-Meier survival curve analysis showed that the overall survival time of CDCA3 positive group was significantly lower than that of CDCA3 negative group(P<0.05).4.Cox proportional hazard regression model analysis showed that the positive expression of CDCA3 was an independent risk factor affecting the overall survival of patients with hypopharyngeal squamous cell carcinoma(P<0.05).Part 2 The effect of knockdown CDCA3 on the biological function of HSCC cells in vitroObjectives:The expression of CDCA3 in hypopharyngeal squamous cell carcinoma FaDu cell line was detected.A lentivirus silenced CDCA3 cell line was constructed to study its effects on cell proliferation,apoptosis,cell migration and invasion and other phenotypes in vitro.Methods:1.The qRT-PCR technique was used to detect the expression of CDCA3 in the FaDu cell line of HSCC and HSC-4 cell line of oral squamous cell carcinoma.2.A lentivirus silenced CDCA3 cell line was constructed.3.qRT-PCR and Western blot were used to detect the expression levels of CDCA3 in different treatment groups to verify the efficiency of CDCA3 knockdown.4.Celigo and MTT assays were used to detect the effect of knocking down CDCA3 on cell proliferation ability.5.The apoptosis and cell cycle changes of hypopharyngeal squamous cell carcinoma cells in different treatment groups were detected by flow cytometry assay.6.The migration and invasion ability of FaDu cell in different treatment groups were detected by tr answell assay.Results:1.qRT-PCR result showed that CDCA3 was highly expressed in FaDu cell line.2.Both qRT-PCR and Western blot analysis showed that the level of CDCA3 in shCDCA3 group were significantly lower than those of shCtrl control group(P<0.05).3.Celigo and MTT assays showed that the cell proliferation ability of shCDCA3 group was suppressed compared to that of shCtrl control group(P<0.05).4.The results of flow cytometry showed that the apoptosis rate of shCDCA3 group was higher than that of shCtrl control group(P<0.05),and cell cycle arrest appeared.5.Transwell assay found that the migration and invasion ability of shCDCA3 group was significantly lower than that of shCtrl control group(P<0.05).Part 3 The molecular mechanism of CDCA3 in HSCCObjectives:The transcriptome of the two groups(shCDCA3 group and of shCtrl control group)were sequenced by next-generation sequencing technology.Then bioinformatics analysis was performed to explore the molecular biological mechanism of the effect of CDCA3 on hypopharyngeal squamous cell carcinoma.Methods:1.The transcriptome of the two groups were sequenced by next-generation sequencing technology.Then bioinformatics analysis was performed to find differentially expressed genes.Through the enrichment analysis of GO and KEGG pathways,the differentially expressed genes were analyzed,and the molecular biological mechanism of the influence of CDCA3 expression on the biological characteristics of hypopharyngeal squamous cell carcinoma cells was explored.2.Western bloting was carried out to verify the changed molecular pathways.Results:1.Bioinformatics analysis results suggested that knockdown of CDCA3 caused changes of AKT/mTOR signaling pathway.2.Western blot analysis results confrmed that CDCA3 knockdown caused down-regulation of the expression of AKT and mTOR.Conclusions1.The expression of CDCA3 increased in hypopharyngeal squamous cell carcinoma tissues and cells.CDCA3 expression level was significantly related with tumor T stage,TNM stage,lymph node metastasis and prognosis.The positive expression of CDCA3 is a risk factor affecting the prognosis of patients.2.The proliferation,migration and invasion ability of FaDu cells are significantly suppressed after CDCA3 knockdown,and the suppression of cell proliferation may be related to cell cycle arrest and increased apoptosis.3.CDCA3 may promote the tumorigenesis and development of hypopharyngeal squamous cell carcinoma through the AKT/mTOR signaling pathway.